Comparison of mobile phone microscopes.

<p>A) <i>Left panel:</i> Cartoon schematic of a ball lens mobile phone microscope. Red brackets indicate microscope attachment optics outside of the phone (a ball lens), and blue brackets indicate mobile phone camera optics inside the phone (a lens group and CMOS sensor). <i>Middle panel:</i> Image of stained cheek epithelial cells taken with a 6 mm ball lens. <i>Right panel:</i> Enlargement of the area indicated within the dashed line in the middle panel. B) <i>Left panel</i>: Cartoon schematic of a standard finite objective microscope attachment to a mobile phone, consisting of an objective and an eyepiece. <i>Middle panel:</i> Image of stained cheek epithelial cells taken with a 4X/0.10 NA objective and a 20X eyepiece. <i>Right panel:</i> Enlargement of the area indicated within the dashed line in the middle panel. Note that despite the image being in-focus at the center of the field of view, some image degradation due to field curvature is detectable at the edge of the field. C) <i>Left panel:</i> Cartoon schematic of the reversed lens microscope presented in this paper, with opposing identical lens groups outside the phone (red brackets) and inside the phone (blue brackets). <i>Middle panel:</i> Image of stained cheek epithelial cells taken with the opposed lens group setup. <i>Right panel:</i> Enlarged area of the area indicated within the dashed line in the middle panel. Note that despite the image being focused at the center of the field, no field curvature is detectable in the reversed lens microscope image, in contrast to the ball lens A) and standard finite objective B) microscope images.</p

Illumination of the reversed lens microscope.

<p>A) Cartoon schematic of the illumination optics together with the collection optics. Red and blue brackets indicate optics outside and inside the phone, respectively. Green brackets indicate the illumination system. A single LED illuminates the sample through an illumination shaping filter (ISF, dashed line) and a diffuser (solid line). B) Methods for correcting image intensity variation caused by vignetting. Columns correspond to the method used. For each column, the top panel is an image of a blank sample showing the illumination uniformity (or lack thereof). The middle panel is a line scan of this image from corner to corner. The lower panel is the standard deviation of a 10×10 pixel box at the indicated positions. Column 1 shows the results of using an LED to directly illuminate the sample. Column 2 shows the results of adding a diffuser between the LED and the sample. Column 3 shows the results of adding illumination shaping filters between the LED and the diffuser. Column 4 shows the results of incorporating a modified form of high-dynamic-range imaging with the diffuser and illumination shaping filters. Images at multiple illumination levels are taken and combined into a single image. Parts of the sample that fall into vignetted regions on the sensor are substituted with the corresponding region of the images taken with brighter illumination levels (see Methods). Note that this image has not yet been flat fielded based on the calibration image. C) An image of a 0.05 mm spacing Ronchi ruling taken with the reversed lens microscope and the combined illumination correction methods described in B). A 10X zoom of a portion of the Ronchi ruling is shown in the upper right corner.</p

Resolution of the reversed lens microscope.

<p>A) Ray-tracing model of a reversed mobile phone camera lens as an objective for a mobile phone microscope. Performance is predicted to be best on axis (α), falling off by >2X at the edge of the field (δ) for a 1.0 mm spacing between lenses. Optical resolution is in microns; to account for variations in sagittal and tangential point-spread at higher field angles, resolution was defined as the first-zero radius of an Airy disk chosen such that its 70% encircled energy radius is the same as that computed for the sample point via ZEMAX. Field positions for α, β, γ, and δ are 0.0, 0.7, 1.5, and 2.1 mm, respectively. B) Measurements of resolution achieved by the reversed lens microscope. The resolution measurements are based on the smallest resolvable group of a 1951 USAF resolution target imaged at different radial distances from the optic axis; asymmetric NA at high field angles (and thus field radii) results in differing sagittal and tangential resolution, as seen in c and d. The dashed line connects to enlargements of the target at the different field positions.</p

A multi-phone mobile microscope.

<p><b>A</b> Diagram of the magnifying optics and illumination added to a mobile phone to create a transmission light microscope. <b>B</b> Prototype of a field-ready mobile microscope – the CellScope – that has a folded optical path for compactness and is equipped with a multi-phone holder and iPhone 4. Phone-specific variants have been evaluated on five continents for various applications. <b>C</b> A Wright stained blood smear taken on the mobile microscope with an iPhone 4 and 20×/0.4 NA objective showing the inscribed field of view captured by the device. <b>D</b> Enlarged images of the small region of interest in <b>C</b> containing a granulocyte and red blood cells taken with four different mobile phones. The images demonstrate resolution, color, and brightness differences among phones.</p

Mobile phones differ from scientific cameras in selection of image capture and processing parameters.

<p><b>A</b> Common core hardware components underlie the capture process of both mobile phone cameras and scientific cameras. <b>B</b> The capture and processing parameters are set directly through the user interface of a scientific camera. <b>C</b> On mobile phones, an intermediate layer assesses the view of the camera in real-time and modifies image acquisition. This simplifies the user interface for traditional point-and-shoot photography but sacrifices the control desired by a scientific user.</p

Spatial resolution of mobile phone microscopy has improved with mobile phone advancement.

<p><b>A</b> The spatial resolution of mobile phone microscopy with iPhone and Android phones is plotted as a function of the effective pixel size for images taken with a 10×/0.25 NA objective. The theoretical constraints on resolution imposed by pixel spacing on the Bayer color sensor array are plotted along with the empirically determined resolution limit of the underlying microscope optics. <b>B</b> The spatial resolution of mobile phone microscopy with the same iPhone and Android phones is plotted as a function of megapixel count. <b>C</b> Spatial resolution of the iPhone family of phones is plotted over time, together with the dates of significant camera advancements.</p

Proposed steps to enable quantitative, reproducible imaging with a mobile phone microscope.

<p><b>A</b> Standardize illumination source and brightness. <b>B</b> Set focal state on a field with known dimensions or features. <b>C</b> Set exposure and gain using a clear field of view. Use this field to set or select a white balance state; may require resetting exposure and gain to ensure changes in white balance do not result in saturation of a color channel. <b>D</b> Acquire images of samples while keeping capture settings constant. <b>E</b> Information content can be preserved by selecting lossless or high quality compression settings. In addition, multiple images can be used to record additional z planes or expand the effective dynamic range of the image. While many of the features required to implement these steps are not directly accessible in the default camera, they are built into commonly available third-party camera applications or can be incorporated into custom applications.</p

Illumination variation, image distortion, and pixel non-linearity of mobile phone microscopy can be minimized.

<p><b>A</b> Variation in illumination across a clear field of view along the horizontal and vertical axes for an LED flashlight source evaluated with a 10×/0.25 NA objective and iPhone 4. <b>B</b> Distortion across a field of view, evaluated along a bar target with a 10×/0.25 NA objective and iPhone 4. A parabolic fit is superimposed on the data for both axes. <b>C</b> The measured pixel response (<b>+</b>) of an iPhone 4 and 10×/0.25 NA objective to changes in illumination intensity is nonlinear but can be corrected for the gamma encoding (<b>o</b>) to recover a linear pixel response (R² = 0.999).</p

Phone automation during image capture and storage degrades feature and color information.

<p><b>A</b> High-contrast image of an array of 20 µm diameter pinholes in chrome taken with an iPhone 4 and 20×/0.40 NA objective. <b>B</b> Phone auto-focus changes effective magnification for samples held at different distances from the objective lens, resulting in changes to apparent feature size. <b>C</b> Saturation in individual color channels due to auto-exposure and gain causes loss of color contrast in sparse, bright samples. <b>D</b> Built-in white-balance of the iPhone 4 is insufficient to overcome variable illumination conditions, resulting in a shift of the apparent color profile of samples such as this blood smear, as quantified in <b>E</b> and <b>F</b> for a region of interest illuminated with a white LED or halogen lamp, respectively. <b>G</b> Sharpening algorithms cause patterns of artificial ringing and haloing around high contrast structures such as this chrome-on-glass resolution target. <b>H</b> Intensity profiles at edges show phone-specific deviations from the expected theoretical monotonic profile obtained from an incoherently illuminated sample using a scientific camera and no image processing. The measured profiles are normalized, with 0 corresponding to the intensity in chrome far from an edge and 1 corresponding to the intensity in a clear region far from an edge.</p

Spatial resolution of mobile phone microscopy is dependent on microscope optics.

<p><b>A</b> The resolution that can be captured with a mobile phone microscope approaches that of a scientific camera coupled to the same optics across a range of numerical apertures. Inset shows the measured intensity profile across bars of non-transmitting chrome spaced at 512 line pairs per millimeter and taken with a 10×/0.25 NA objective, as well as the ideal target profile. The Michelson contrast calculated for this example group is 41%, indicating that features with this spacing are resolved. <b>B</b> Wright stained blood smear with an inset of a granulocyte and red blood cells taken with a 10×/0.25 NA objective and iPhone 4. <b>C</b> Image of the same sample and region of interest taken with a 40×/0.65 NA objective and iPhone 4 showing improved resolution.</p